Anaplastic Lymphoma Kinase Overexpression Research Articles

Case report: ALK-positive histiocytosis presented as bilateral synchronous breast masses with long-term remission on crizotinib

ALK-positive histiocytosis (APH) is a rare type of histiocytic neoplasm with characteristic ALK (Anaplastic Lymphoma Kinase) gene translocation and fusion, with only 27 reported cases in the literature. In this study, we report the first case of synchronous bilateral breast involvement of ALK-positive histiocytosis on initial presentation in a 46-year-old Hispanic woman. APH was diagnosed by the confirmation of clonal histiocyte proliferation with ALK overexpression on IHC and the presence of KIF5B-ALK gene fusion from her breast and lung biopsies. The patient in our study is currently under complete and long-term remission with crizotinib treatment (an ALK inhibitor). This report expands on the clinical manifestation of APH, emphasizes the importance of ALK detection in histiocytic diseases, and provides the efficacy and long-term prognosis of the ALK inhibitor therapy for APH.

Anaplastic lymphoma kinase overexpression enhances aggressive phenotypic characteristics of endometrial carcinoma

BackgroundAlthough anaplastic lymphoma kinase (ALK) is overexpressed in several primary solid tumor types, its role in endometrial carcinoma (Em Ca) remains unclear.MethodsWe evaluated expression of ALK and its related molecules in clinical samples consisting of 168 Em Ca tissues. We also used Em Ca cell lines to evaluate the functional role of ALK.ResultsCytoplasmic ALK immunoreactivity in the absence of chromosomal rearrangement was positively correlated with ALK mRNA expression, and was significantly higher in Grade (G) 3 Em Ca than in G1 or G2 tumors. ALK immunoreactivity was also significantly associated with expression of cancer stem cell (CSC)-related molecules (cytoplasmic CD133, ALDH1, Sox2) and neuroendocrine markers (CD56 and synaptophysin). Although the proliferative index was significantly higher in ALK-positive Em Ca when compared to ALK- negative malignancies, there was no association between ALK expression and other clinicopathological factors in this disease. In Em Ca cell lines, full-length ALK overexpression increased proliferation, decreased susceptibility to apoptosis, enhanced cancer stem cell features, and accelerated cell mobility, whereas these phenotypes were abrogated in ALK-knockdown cells. Finally, patients with tumors harboring either wild-type ALK or high ALK mRNA expression had a poorer prognosis than those with either mutant ALK or low ALK mRNA expression.ConclusionFull-length ALK overexpression occurs in a subset of Em Ca, particularly in G3 tumors, and contributes to the establishment and maintenance of aggressive phenotypic characteristics through modulation of several biological processes.

New perspectives for targeting therapy in ALK-positive human cancers.

Anaplastic lymphoma kinase (ALK) is a member of the insulin receptor protein-tyrosine kinase superfamily and was first discovered in anaplastic large-cell lymphoma (ALCL). ALK alterations, including fusions, over-expression and mutations, are highly associated with cancer initiation and progression. This kinase plays an important role in different cancers, from very rare to the more prevalent non-small cell lung cancers. Several ALK inhibitors have been developed and received Food and Drug Administration (FDA) approval. However, like other drugs used in targeted therapies, ALK inhibitors inevitably encounter cancer cell resistance. Therefore, monoclonal antibody screening based on extracellular domain or combination therapies may provide viable alternatives for treating ALK-positive tumors. In this review, we discuss the current understanding of wild-type ALK and fusion protein structures, the pathological functions of ALK, ALK target therapy, drug resistance and future therapeutic directions.

Systematic identification of ALK substrates by integrated phosphoproteome and interactome analysis.

The sensitivity of phosphorylation site identification by mass spectrometry has improved markedly. However, the lack of kinase-substrate relationship (KSR) data hinders the improvement of the range and accuracy of kinase activity prediction. In this study, we aimed to develop a method for acquiring systematic KSR data on anaplastic lymphoma kinase (ALK) using mass spectrometry and to apply this method to the prediction of kinase activity. Thirty-seven ALK substrate candidates, including 34 phosphorylation sites not annotated in the PhosphoSitePlus database, were identified by integrated analysis of the phosphoproteome and crosslinking interactome of HEK 293 cells with doxycycline-induced ALK overexpression. Furthermore, KSRs of ALK were validated by an in vitro kinase assay. Finally, using phosphoproteomic data from ALK mutant cell lines and patient-derived cells treated with ALK inhibitors, we found that the prediction of ALK activity was improved when the KSRs identified in this study were used instead of the public KSR dataset. Our approach is applicable to other kinases, and future identification of KSRs will facilitate more accurate estimations of kinase activity and elucidation of phosphorylation signals.

Comprehensive assessment of anaplastic lymphoma kinase in localized and metastatic prostate cancer reveals targetable alterations.

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase with genomic and expression changes in many solid tumors. ALK inhibition is first line therapy for lung cancers with ALK alterations, and an effective therapy in other tumor types, but has not been well-studied in prostate cancer. Here, we aim to delineate the role of ALK genomic and expression changes in primary and metastatic prostate cancer. We determined ALK expression by immunohistochemistry and RNA-Seq, and genomic alterations by NGS. We assessed functional consequences of ALK overexpression and pharmacological ALK inhibition by cell proliferation and cell viability assays. Among 372 primary prostate cancer cases we identified one case with uniformly high ALK protein expression. Genomic analysis revealed a SLC45A3-ALK fusion which promoted oncogenesis in in vitro assays. We observed ALK protein expression in 5/52 (9%) of metastatic prostate cancer cases, of which 4 of 5 had neuroendocrine features. ALK-expressing neuroendocrine prostate cancer had a distinct transcriptional program, and earlier disease progression. An ALK-expressing neuroendocrine prostate cancer model was sensitive to pharmacological ALK inhibition. In summary, we found that ALK overexpression is rare in primary prostate cancer, but more frequent in metastatic prostate cancers with neuroendocrine differentiation. Further, ALK fusions similar to lung cancer are an occasional driver in prostate cancer. Our data suggest that ALK-directed therapies could be an option in selected patients with advanced prostate cancer.

Integrated analysis of ALK higher expression in human cancer and downregulation in LUAD using RNA molecular scissors.

Anaplastic lymphoma kinase (ALK) is an endorsed molecular target in ALK-rearranged carcinomas, including lung adenocarcinoma. However, the clinical advantage of targeting ALK using druggable inhibitors is almost universally restricted by the development of drug resistance. Therefore, a strategy for combating ALK overexpression remains paramount for ALK-driven cancer. We systemically analyzed the overexpression pattern of ALK and its clinical consequences, genetic alterations, and their significance in cancer hallmark genes, and correlation using integrated multidimensional approaches. The LwCas13a RNA molecular scissors was used to downregulate ALK-rearrangement by leveraging two target guide RNAs in lung adenocarcinoma (LUAD) cells. Immunocytochemistry, immunoblotting, and MTT assays were conducted to validate the downregulation. We found elevated levels of ALK in several malignancies, including LUAD, than in normal tissues. Higher expression of ALK was significantly associated with worse or shorter survival than patients with lower expression. We identified numerous genetic alterations in ALK, which potentially alter the cancer hallmark genes, including STAT1 and CTSL, in patients with LUAD. Next, we observed that the LwCas13a molecular scissors robustly downregulated both phosphorylated and total ALK chimera protein expression in LUAD cells compared to the control. Furthermore, we found that downregulation of ALK chimera protein substantially inhibited cell viability and induced cell death, including apoptosis. Our findings suggest a basis for ALK as a prognostic biomarker and the LwCas13a molecular scissors successfully downregulated the onco-driver ALK-rearrangement protein, which will potentially pave the way toward the development of novel therapeutic strategies for ALK-driven cancer.

Immunohistochemical Expression of Anaplastic Lymphoma Kinase in Invasive Breast Carcinomas- A Retrospective Study

Introduction: Breast carcinoma remains one of the most common causes of mortality among female cancer patients in spite of improvements in treatment modalities. Increased survival rate can be achieved by identification of new targeted therapies. Anaplastic Lymphoma Kinase (ALK) alterations are present in many solid and hematological tumours indicating its role in pathogenesis and treatment. There are studies regarding the expression of ALK in few breast cancers but its importance was not clearly mentioned. Hence, identification of ALK overexpression in breast cancers, particularly in Triple Negative Breast Cancers (TNBC) might play a role in their chemotherapy with the help of ALK inhibitors. Aim: To study the ALK expression in different subtypes of invasive carcinomas of breast. Materials and Methods: This was a retrospective crosssectional study conducted in the Department of Pathology at Great Eastern Medical School (GEMS) & Hospital, Srikakulam, Andhra Pradesh, India, from January 2022 to July 2022. The data of 60 patients, from January 2021 to December 2021 was retrieved using Hospital Information Management System (HIMS) and the Hematoxylin & Eosin-stained slides and formalin fixed paraffin embedded tissue blocks of breast tumour were retrieved and reviewed. Estrogen receptor (ER), Progesterone receptor (PR) and Human Epidermal growth factor Receptor 2 (HER2) immunostains were performed and categorized based on molecular classification as Luminal, Her2 and Triple negative. ALK Staining was performed on all cases and its expression was studied. Statistical Package for Social Sciences (SPSS) software version 2.0 was used for analysis of data. Mean with standard deviation is used for quantitative variables and prevalence, ratio is used for quantitative variables. Chi-square test and Fischer exact test were used for detecting significance. The p-value <0.05 was considered as significant. Results: Out of 60 cases, majority(N=22;36.66%) of patients were in the age group of 51 to 60 years. The mean tumour size was 3.2±0.5 cm. The most common histological type was invasive breast carcinoma, No Special Type (N=44;73.34%). Majority of the tumours showed grade 1 and 2 with 24 (40%) and 25 (41.67%) cases respectively. A total of 30 cases (50%) of tumours belong to stage T2. Luminal molecular subtype is the most common 31 cases (51.67%) followed by TNBC, 16 cases(26.67%) and Her2neu 13 cases (21.67%). Among all the cases, ALK overexpression was seen in 17 (28.33%) cases and among different molecular subtypes, its expression is seen in 5 (8.33%) cases of Luminal type, 3 (5.0%) cases of HER2 type and in 9 (15.0%) cases of TNBC cases. Conclusion: Immunohistochemical analysis showed ALK over expression in a substantial proportion of cases and possibly plays a significant role in aggressive behaviour of breast cancer. ALK inhibitors offer an opportunity to treat aggressive subtypes of breast cancer.

ALK Protein Expression Patterns in Squamous Cell Carcinoma of the Oral Cavity.

Oral squamous cell carcinoma (OSCC) is characterized by a broad spectrum of genomic imbalances, including gross chromosomal (polysomy/aneuploidy) ones as well as specific gene alterations. Aberrant expression of anaplastic lymphoma kinase (ALK) seems to be a useful molecular marker for discriminating patients based on genetic signatures in a variety of solid malignancies, such as lung carcinoma. Our aim was to analyze ALK protein expression patterns in a series of OSCCs. Fifty (n=50) OSCC tissue sections were analyzed by implementing an ALK-based immunohistochemistry protocol. Digital image analysis was performed for measuring the corresponding protein expression levels. ALK overexpression was observed in 14/50 (28%) OSCC tissue sections, whereas the rest 36/50 (72%) demonstrated low expression levels. ALK expression was negatively associated with grade (p=0.027) and stage (p=0.0028) of the examined cases. Abnormal ALK expression in subsets of patients with OSCC seems to be related to an aggressive phenotype (advanced stage/progressive dedifferentiation). ALK protein overexpression may be used as a significant marker for applying targeted therapeutic regimens.

Anaplastic Lymphoma Kinase Overexpression Is Associated with Aggressive Phenotypic Characteristics of Ovarian High-Grade Serous Carcinoma

Deregulated full-length anaplastic lymphoma kinase (ALK) overexpression has been found in some primary solid tumors, but little is known about its role in ovarian high-grade serous carcinoma (HGSC). The current study focused on the functional roles of ALK in HGSC. Cytoplasmic ALK immunoreactivity without chromosomal rearrangement and gene mutations was significantly higher in HGSC compared with non-HGSC-type ovarian carcinomas, and was significantly associated with several unfavorable clinicopathologic factors and poor prognosis. HGSC cell lines stably overexpressing ALK exhibited increased cell proliferation, enhanced cancer stem cell features, and accelerated cell mobility, whereas these phenotypes were abrogated in ALK-knockdown cells. Expression of the nervous system-associated gene, ELAVL3, and the corresponding protein (commonly known as HuC) was significantly increased in cells overexpressing ALK. Expression of SRY-box transcription factor (Sox)2 and Sox3 (genes associated with the neural progenitor population) increased in ALK-overexpressing but not ALK-knockdown cells. Furthermore, overexpression of Sox2 or Sox3 enhanced both ALK and ELAVL3 promoter activities, suggesting the existence of ALK/Sox/HuC signaling loops. Finally, ALK overexpression was attributed to increased expression of neuroendocrine markers, including synaptophysin, CD56, and B-cell lymphoma 2, in HGSC tissues. These findings suggest that overexpression of full-length ALK may influence the biological behavior of HGSC through cooperation with ELAVL3 and Sox factors, leading to the establishment and maintenance of the aggressive phenotypic characteristics of HGSC.

Inflammatory myofibroblastic tumor: A multi-institutional study from the pediatric surgical oncology research collaborative.

10024 Background: Inflammatory myofibroblastic tumor (IMT) is a mesenchymal neoplasm of intermediate malignancy, predominantly seen in children and young adults. Given its rarity, data are limited. We describe the largest cohort of patients with IMT to date, with an aim to further characterize this poorly understood tumor. Methods: A multi-institutional review was performed at 18 North American hospitals participating in the Pediatric Surgical Oncology Research Collaborative to identify IMT patients ≤39 years, diagnosed from 2000-2018. Descriptive statistics are described as median and interquartile range. Multivariable analysis was used to identify predictors of event free survival (EFS). Results: 182 patients were identified with a median age of 11 years (5-17); 52% were female. 33% of tumors were thoracic, 26% abdominal/pelvic, 20% head/neck, and 14% genitourinary. Common presenting symptoms included pain (29%), respiratory symptoms (24%), weight loss (12%), and a palpable mass (10%). Median tumor size was 3.9 cm (1.9-6.5). Anaplastic lymphoma kinase (ALK) overexpression was identified via immunohistochemistry in 53% of patients tested. One third of patients had abnormal cytogenetics, with 12% of the entire cohort having an ALK mutation. 7% of patients had distant disease at diagnosis. 13% of patients received neoadjuvant therapy including chemotherapy (3%), ALK inhibitor (4%), radiation (0.5%), non-steroidal anti-inflammatories (NSAIDs) (7%), or steroids (2%). Of those who underwent resection with known margin status (n = 158), 66% had complete resection, 20% had microscopic positive margins, and 14% had gross residual disease. Just over 40% of patients had an en bloc resection of involved organs, most commonly lung (26%). 21% of patients received adjuvant therapy, including chemotherapy (3%), ALK inhibitor (9%), radiation (0.5%), NSAIDs (8%), or steroids (5%). 12% of all patients received an ALK inhibitor: 24% neoadjuvant, 62% adjuvant, and 14% without surgery. Median follow-up time was 36 months (14-69). Overall 5-year survival (OS) was 95% and 5-year EFS was 80%. Predictors of recurrence included respiratory symptoms, larger tumor size, or distant disease at diagnosis. Gender, race, age and primary site were not predictive of EFS. Likewise, there was no association of ALK overexpression or ALK mutation with EFS. The presence of gross or microscopic margins following resection was not associated with recurrence. Conclusions: IMT is a rare tumor with favorable OS. Five year recurrence rate was 20%. Presenting with respiratory symptoms, larger tumor size, or metastatic disease was associated with recurrence, while ALK positivity was not. Notably, a positive margin after resection was not associated with increased recurrence, indicating that aggressive attempts at surgical resection that would compromise form or function may not be warranted.

Inflammatory Myofibroblastic Tumor After Lung Transplant—A Rare and Aggressive Complication: A Case Report

IntroductionMalignant diseases are well-known complications after lung transplantation (LT). Among these, inflammatory myofibroblastic tumor (IMT) is a rare neoplasm with a not well-known and often aggressive biological behavior. Material and MethodsWe hereby describe 2 cases of cystic fibrosis patients who underwent bilateral sequential LT (BSLT) complicated by IMT. ResultsA 26-year-old man presented a right endobronchial lesion 6 months after BSLT. Two consecutive fiber bronchoscopic biopsies showed granulation tissue. For the persistent lesion growth, the patient underwent a transthoracic biopsy showing histologic diagnosis of IMT. Therefore, he underwent to right pneumonectomy that was unfortunately complicated after 6 months with a late bronchopleural fistula and empyema with exitus 6 months later. A 31-year-old woman 1 year after BSLT presented with a left voluminous pleural-parenchymal lesion; the histologic examination after biopsy revealed an IMT. She underwent a removal of the lesion with a macroscopic R0 resection. Histologic, immunophenotypic, and cytogenetic examinations showed a strong overexpression of anaplastic lymphoma kinase requiring biological adjuvant therapies; however, the patient refused it. Four years later, she presented a recurrence treated with debulking procedure and adjuvant radiotherapy. At last follow-up, the patient was alive with stable disease and optimal graft function. ConclusionsAlthough IMT is a rare complication after lung transplant, to obtain a careful diagnosis, an early and aggressive treatment is mandatory.

Analysis of ALK, MYCN, and the ALK ligand ALKAL2 (FAM150B/AUGα) in neuroblastoma patient samples with chromosome arm 2p rearrangements.

Gain of chromosome arm 2p is a previously described entity in neuroblastoma (NB). This genomic address is home to two important oncogenes in NB-MYCN and anaplastic lymphoma kinase (ALK). MYCN amplification is a critical prognostic factor coupled with poor prognosis in NB. Mutation of the ALK receptor tyrosine kinase has been described in both somatic and familial NB. Here, ALK activation occurs in the context of the full-length receptor, exemplified by activating point mutations in NB. ALK overexpression and activation, in the absence of genetic mutation has also been described in NB. In addition, the recently identified ALK ligand ALKAL2 (previously described as FAM150B and AUGα) is also found on the distal portion of 2p, at 2p25. Here we analyze 356 NB tumor samples and discuss observations indicating that gain of 2p has implications for the development of NB. Finally, we put forward the hypothesis that the effect of 2p gain may result from a combination of MYCN, ALK, and the ALK ligand ALKAL2.

Combined EGFR/ALK Expression Analysis in Laryngeal Squamous Cell Carcinoma.

Epidermal growth factor receptor (EGFR) acts as an oncogene in malignancies. Our aim was to examine the role of combined EGFR/ anaplastic lymphoma kinase (ALK) expression as molecular markers in laryngeal squamous cell carcinoma (LSCC) patients. Fifty (n=50) tissue sections derived from twenty-five (n=25) primary LSCCs were analyzed by immunohistochemistry (IHC). EGFR overexpression was observed in 17/25 (68%) cases. Concerning ALK, 23/25 (92%) demonstrated low expression. EGFR expression was associated with grade (p=0.049), whereas ALK expression was correlated with stage (p=0.048). ALK overexpression was detected at advanced-stage EGFR-positive cases. A biphasic EGFR protein expression pattern was observed in five (n=5) LSCC cases, whereas ALK expression was stable in all cases. EGFR overexpression is frequently observed in LSCC combined with low ALK expression. LSCC patients with EGFR/ALK protein overexpression should be eligible for targeted therapeutic strategies.

Abstract 5347: Somatic mutation and overexpression of anaplastic lymphoma kinase (ALK) are uncommon events in Asian head and neck cancers

Abstract Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that is frequently translocated in anaplastic large cell lymphoma (ALCL) and non-small cell lung cancer (NSCLC), etc. Gain-of-function mutations, largely mutations associated with resistance to ALK inhibitors, have been identified in neuroblastoma, colon adenocarcinoma and prostate adenocarcinoma, etc. Pan-cancer analysis of whole-exome sequencing data of >25 cancer types showed a mutational rate ranging from 0-9.4% across pan-cancers (www.cbioportal.org). In cancers of the head and neck, the potential role of ALK on precision treatment has been demonstrated in an Asian head and neck sarcomatoid carcinoma patient bearing an ALK aberration (Kim et al., 2015). In this study, we sought to examine the mutational rates of ALK in the US-TCGA HNSCC Provisional cohort, in a small Asian HNSCC cohort, as well as in Nasopharyngeal carcinoma (NPC, an Asian-prevalent HNC). Analysis of the whole-exome sequencing (WES) data of the US-TCGA HNSCC Provisional cohort (N=527; largely Caucasian ethnicity, 85.6%; www.cbioportal.org) revealed a somatic mutation rate of 4.0% (21 out of 527 patients, with 18 non-synonymous and 3 synonymous mutations) and a gene amplification rate of 0.4% (2 out of 522 tumors with copy number change data available). In contrast, the Asian prevalent Epstein-Barr Virus-associated NPC (N=102 micro-dissected tumors with paired blood samples analyzed by WES), no somatic ALK mutation was identified. Using a small Asian HNSCC cohort (N=16), next-generation sequencing (NGS) analysis with an ALK exon coverage of ~90% revealed no somatic ALK mutation, but the presence of Asian-predilected SNPs with unknown clinical or biological significance. Lastly, ALK protein expression, analyzed by Western blotting, was found to be low or largely undetectable in 25 Asian HNSCC tumors analyzed thus far. Yet, we cannot exclude the possibility of focal ALK expression in HNSCC tumors, which can be largely obscured by Western blotting. In conclusion, somatic mutation of ALK appears to be rare events in Asian HNSCC and EBV-associated Nasopharyngeal carcinoma. Acknowledgements: VWYL receives funding supports from the Research Grant Council, Hong Kong (General Research Fund: #1711484, #17121616; Theme-based Research: T12-401/13-R) and the Hong Kong Cancer Fund, Hong Kong. KWL receives funding from the Research Grant Council (Theme-based Research: T12-401/13-R), Hong Kong. WP is supported by Faculty Postdoctoral Fellowship Scheme from the Faculty of Medicine, Chinese University of Hong Kong. JYKC receives funding support by the Dr Stanley Ho Medical Foundation. Reference Kim, S. M., Kim, M. J., Jung, H. A., Sun, J. M., Choi, Y. L., Ko, Y. H., . . . Ahn, M. J. (2015) Presence of anaplastic lymphoma kinase translocation in sarcomatoid carcinoma of head and neck and treatment effect of crizotinib: A case series. Head Neck, 37(5), E66-69 Citation Format: Wenying Piao, Peony Hiu Yan Poon, Lan Wang, Chin Wang Lau, Jason Ying Kuen Chan, Yuxiong Su, Amy Bik Wan Chan, Hoi Lam Ngan, Kwok Wai Lo, Vivian Wai Yan Lui. Somatic mutation and overexpression of anaplastic lymphoma kinase (ALK) are uncommon events in Asian head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5347.

A digital PCR based assay to detect all ALK fusion species

Accurate detection of Anaplastic lymphoma kinase (ALK) fusion in lung cancer cells is essential to screen for patients suitable for targeted drug treatments such as crizotinib. ALK fusion involves multiple genes and different exon junctions. When an ALK fusion results in overexpression of the ALK protein, a malignant transformation can occur. The challenge is to develop a single test that will detect ALK overexpression from all fusion combinations including novel/unidentified fusion partner(s). In this study, we evaluated two strategies for detecting ALK fusion events using digital PCR: 5′/3′ imbalance and ALK overexpression relative to a reference gene. Our data shows that the determination of ALK RNA expression levels is a better option when a reference gene is included in the assay. We further determined the threshold to call for positive or negative samples and evaluated the analytical specifications of the assay in 28 FFPE samples from Non-small cell lung cancer (NSCLC) patients with know ALK fusion status. We validated this threshold with 36 clinical samples with ALK status determined by IHC. Digital PCR ALK assay we developed had a concordance of 97.2% (35/36). Testing the assay on clinical samples demonstrated consistency with reference assays, suggesting a great potential for the dPCR assay to service the clinical detection needs.

ALK signaling cascade confers multiple advantages to glioblastoma cells through neovascularization and cell proliferation.

Anaplastic lymphoma kinase (ALK), which is a receptor tyrosine kinase, is essentially and transiently expressed in the developing nervous system. Here we examined the functional role of the ALK gene in glioblastomas (GBMs). In clinical samples of GBMs, high ALK expression without gene rearrangements or mutations was frequently observed in perivascular lesions, in contrast to the relatively low expression in the perinecrotic areas, which was positively correlated with N-myc and phosphorylated (p) Stat3 scores and Ki-67 labeling indices. ALK immunoreactivity was also found to be associated with neovascular features including vascular co-option and vascular mimicry. In astrocytoma cell lines, cells stably overexpressing full-length ALK showed an increase in expression of pStat3 and pAkt proteins, as well as hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor-A (VEGF-A) mRNAs, in contrast to cells with knockdown of endogenous ALK which showed decreased expression of these molecules. Transfection of the constitutively active form of Stat3 induced an increase in HIF-1α promoter activity, and the overexpression of HIF-1α in turn resulted in enhancement of VEGF-A promoter activity. In addition, cells with overexpression or knockdown of ALK also showed a tendency toward increased and decreased proliferation, respectively, through changes in expression of pAkt and pStat3. Finally, ALK promoter was significantly activated by transfection of Sox4 and N-myc, which are known to contribute to neuronal properties. These findings therefore suggest that N-myc/Sox4-mediated ALK signaling cascades containing Stat3, Akt, HIF-1α, and VEGF-A confer multiple advantages to tumor growth through alterations in neovascularization and cell proliferation in GBMs.

NPM1‐ALK overexpression‐driven toxicity in ALCL is partner dependent and driven by phosphorylation of novel substrates leading to overstimulation of biosynthetic pathways

Introduction: Activation of the tyrosine-kinase domain of anaplastic lymphoma kinase (ALK) drives malignant growth in subsets of several cancers as part of oncogenic fusions resulting from chromosomal rearrangements. ALK stimulates several oncogenic pathways, including JAK/STAT3, MEK/ERK, and PI3K/AKT. Clinically, 70% of anaplastic large-cell lymphoma (ALCL) and ~5% of nonsmall-cell lung cancer (NSCLC) are ALK+. ALK tyrosine-kinase inhibitors (TKIs) are approved for treatment of NSCLC and are under investigation in ALCL. Clinical resistance in lung cancer typically develops within 10 to 12 months is due to, most commonly, activation of alternate pathways or second-site kinase-domain mutations. We and others previously reported that, unlike NSCLC, ALCL tumor cells have great difficulty finding alternate pathways to replace ALK and instead upregulate expression of the NPM1-ALK fusion to maintain survival during inhibitor exposure. Strikingly, we found a toxic overdose of ALK signaling when inhibitors are withdrawn from overexpressing cells, triggering death. Here, we explore the mechanisms by which ALK overactivation drives cellular toxicity. Materials and Methods: We have generated multiple models of ALK+ ALCL with resistance to all generations ALK inhibitors. We employed RNAseq and phosphoproteomics to reveal candidate drivers of death upon ALK overexpression. We have generated multiple variant alleles of NPM1-ALK that enable us to interrogate the role of ALK's fusion partner in mediating these effects. Results: All generations of ALK inhibitors drive NPM1-ALK overexpression as the most frequent resistance mechanism in ALCL, but increased ALK activity upon inhibitor withdrawal is universally toxic. Extensive interrogations with targeted inhibitors and genetic approaches revealed that none of ALK's well-characterized downstream pathways is a key driver of overdose toxicity. Interestingly, while overexpression of NPM1-ALK confers selective disadvantage, elevated levels of activated ALK kinase domain alone do not. RNAseq analysis reveals hyperstimulation of protein biogenesis and RNA processing as cells die due to NPM1-ALK overdose. Phosphoproteomics results align with RNAseq data showing increased phosphorylation of proteins driving the same pathways. Taken together our results show the NPM1 translocation partner plays a key role driving NPM1-ALK overdose in ALCL by promoting phosphorylation of novel substrates. Conclusions: In contrast to ALK+ NSCLC, ALCL cannot easily bypass a need for ALK by activating alternate pathways. NPM1-ALK overexpression-driven toxicity is partner dependent, driving death associated with hyperactivation of protein and RNA processing pathways. Drugs that drive ALK toward phosphorylation of noncanonical substrates therefore represent a novel therapeutic strategy worth pursuing in ALCL and other ALK-driven malignancies. Keywords: ALK; anaplastic large cell lymphoma (ALCL)

Absence of Chromosomal Translocations and Protein Expression of ALK in Sinonasal Adenocarcinomas

IntroductionChromosomal translocations at 2p23 cause overexpression of anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase involved in signalling pathways that regulate cell proliferation. This translocation occurs in 5% of lung adenocarcinoma and has been demonstrated to be useful as a therapeutic target for crizotinib. Sinonasal adenocarcinomas (SNAC) are histologically similar to lung adenocarcinomas; the aim of this study was to evaluate the presence of ALK alterations in SNAC. MethodBreak-apart fluorescent in situ hybridisation was used to analyse the presence of ALK translocations in 96 tumour samples. In addition, ALK protein expression was studied by immunohistochemistry. ResultsThe samples of SNAC did not show ALK translocation. Moreover, ALK protein expression was absent in all cases. ConclusionsThese results suggest that ALK is not involved in SNAC.

Anaplastic lymphoma kinase expression and gene alterations in glioblastoma: correlations with clinical outcome

AimsTo study anaplastic lymphoma kinase (ALK) protein expression and possible underlying gene alterations in glioblastoma (GBM), correlating them with clinical outcome.MethodsWe studied ALK immunohistochemical expression and fluorescent in situ hybridisation...

A malignant inflammatory myofibroblastic tumor of the hypopharynx harboring the 3a/b variants of the EML4-ALK fusion gene

Inflammatory myofibroblastic tumors (IMT) in the head and neck region are rare neoplasms that generally mimic benign/low-grade neoplasms. Overexpression of anaplastic lymphoma kinase (ALK) has been reported in 50% of IMT cases, secondary to ALK activation by structural rearrangements in the ALK gene, which results in a fusion protein with echinoderm microtubule associated protein like 4 (EML4) in ~20% of cases. The present study describes a case of a 74-year-old woman with a malignant IMT in the right posterior hypopharynx harboring a previously unreported chromosomal rearrangement resulting in EML4 and ALK gene fusion. Strong ALK immunoreactivity was observed in neoplastic cells, while fluorescent in situ hybridization combined with fluorescent fragment analysis and direct sequencing identified the first case of the 3a/b variants of the EML4-ALK fusion gene in IMT. The results of the current study highlight the uncommon occurrence of ALK-positive IMT in the head/neck region and demonstrate the importance of integrating different molecular methodologies to identify unequivocal gene fusion characterization.